Treatment of malaria with farnesyl protein transferase inhibitors

ABSTRACT

Disclosed is a method of treating Malaria comprising administering an effective amount of an FPT inhibitor to a patient in need of such treatment alone or in combination with an additional antimalarial agent and/or agent for reversing antimalarial resistance.  
     Also disclosed are novel Farnesyl Protein Transferase inhibitors.

REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. ProvisionalApplication Serial No. 60/263,277 filed Jan. 22, 2001.

FIELD OF THE INVENTION

[0002] The present invention relates to a method of treating malariacomprising administering an effective amount of an FPT inhibitor to apatient in need of such treatment alone or in combination with anadditional antimalarial agent and/or agent for reversing antimalarialresistance.

[0003] The present invention also relates to novel Farnesyl ProteinTransferase inhibitors, methods of preparing such inhibitors,compositions containing such inhibitors and methods of using suchinhibitors in the treatment of malaria.

BACKGROUND

[0004] Malaria is one of the most widespread infectious diseases in theworld infecting over 200 million people of which, it has been estimated,1-2 million die from the disease per year.

[0005] Malaria is a disease caused by a parasite transmitted by the biteof an infected female Anopheles mosquito. When an infecting sporozoiteparasite enters the bloodstream it rapidly infects both liver and redblood cells and differentiates into merozoites. Asexual reproduction ofthe merozoite within erythrocytes results in the rupture and subsequentreinfection of other red blood cells. This cyclic process results inclinical symptoms of headaches, sweating, vomiting, malaise, deliriumand acute fever and may be fatal if not treated.

[0006] There are four main species, which infect humans, Plasmodiumvivax, Plasmodium malariae, Plasmodium ovale, and Plasmodiumfaliciparum. Several anitmalarial drugs have been developed to treat thedisease. Chloroquine and quinine have been used widely for over fortyyears because they have been exceptionally safe, inexpensive andeffective drugs for treating the disease. Recently, however,chloroquine- and quinine-resistant strains of P. faliciparum havedeveloped and are causing high levels of mortality. In an effort toidentify a new and more effective method for treating malaria,Chakrabarti et. al., have studied the use of inhibitors of prenyltransferases from the malaria parasite, Plasmodium falciparum, in aneffort to identify a method for treating malaria, see Chakrabarti, etal., Molecular and Biochemical Parasitology (1998) 94, 175-184.

[0007] In view of the need to find new treatments of malaria, thoseskilled in the art would welcome an effective method for the treatmentof malaria. The present invention provides such a method utilizinginhibitors of Plasmodium falciparum protein prenyl transferases.

SUMMARY OF THE INVENTION

[0008] This invention provides a method of treating and/or preventingmalaria comprising administering to a patient, in need of suchtreatment, an effective amount (e.g., a therapeutically effectiveamount, or an amount to inhibit malaria) of a Farnesyl ProteinTransferase inhibitor selected from Compounds 1-26 described below.

[0009] This invention also provides novel compounds (i.e., novel FPTinhibitors) selected from the compounds: 14, or 16-23 described below.

DETAILED DESCRIPTION

[0010] As used herein, the following terms are used as defined belowunless otherwise indicated:

[0011] MH⁺-represents the molecular ion plus hydrogen of the molecule inthe mass spectrum;

[0012] BOC-represents-t-BOC-represents-tert-butyloxycarbonyl;

[0013] CBZ-represents —C(O)OCH₂C₆H₅ (i.e., benzyloxycarbonyl);

[0014] CH₃CN-represents-acetonitrile;

[0015] CDCl₃-represents-deuterated chloroform

[0016] CH₂Cl₂-represents dichloromethane;

[0017] CIMS-represents chemical ionization mass spectroscopy;

[0018] DEA-represents diethyl amine;

[0019] DMSO-represents dimethyl sulfoxide

[0020] DMF-represents N,N-dimethylformamide;

[0021] EI-represents electron ionization spectroscopy;

[0022] Et-represents ethyl;

[0023] Et₂O-represents diethyl ether;

[0024] EtOAc-represents ethyl acetate;

[0025] EtOH-represents ethanol;

[0026] HCl-represents hydrochloric acid

[0027] IPA-represents isopropanol;

[0028] LAH-represents lithium aluminum hydride;

[0029] LCMS-represents liquid chromatography mass spectroscopy;

[0030] Me-represents methyl;

[0031] MeOH-represents methanol;

[0032] MgSO₄-represents magnesium sulfate;

[0033] MS-represents mass spectroscopy;

[0034] FAB-represents FABMS which represents fast atom bombardment massspectroscopy;

[0035] HRMS-represents high resolution mass spectroscopy;

[0036] NaOH-represents sodium hydroxide;

[0037] Na₂SO₄-represents sodium sulfate;

[0038] NaHCO₃-represents sodium bicarbonate;

[0039] NH₄OH-represents amonium hydroxide;

[0040] NOE-represents nuclear Overhauser effect;

[0041] NMR-represents nuclear magnetic resonance spectroscopy;

[0042] NMM-represents N-methylmorpholine;

[0043] p-TosCl-represents p-toluenesulfonyl chloride;

[0044] P₂O₅-represents phosphorous pentoxide;

[0045] Pr-represents propyl;

[0046] Et₃N-represents TEA which represents triethylamine;

[0047] t-BUTYL-represents —C—(CH₃)₃;

[0048] TFA-represents trifluoroacetic acid;

[0049] THF-represents tetrahydrofuran;

[0050] TLC-represents thin layer chromatography;

[0051] FPT-represents Farnesyl Protein Transferase

[0052] One skilled in the art will appreciate that the positions of theNitrogen atoms around the imidazole portion of the compounds describedbelow are:

[0053] The FPT inhibitors useful in the claimed invention are: Cmpd. #STRUCTURE 1

see WO 00/37459 2

see WO 00/37459 3

see U.S. Pat. No. 5,874,442 4

see WO 00/37459 5

see WO 00/37458 6

see WO 00/37459 7

see WO 00/37459 8

see WO 00/37459 9

see WO 00/37458 10

see U.S. Pat. No. 5,089,496 11

see U.S. Pat. No. 5,874,442 12

see WO 98/11098 13

see Example 9, below 14

see Example 10, below 15

see WO 98/57960 16

see Example 5, below 17

see Example 6, below 18

see Example 7, below 19

see Example 8, below 20

see Example 3, below 21

see Example 1, below 22

see Example 2, below 23

see Example 4, below 24

See Example 11 below 25

see WO 97/16443 26

see WO 97/30992

[0054] Preferably the compound (3)

[0055] is used in the methods of this application.

[0056] The novel FPT inhibitory compounds are: Cmpd. # STRUCTURE 14

16

17

18

19

20

21

22

23

[0057] In another embodiment, this invention provides a method oftreating malaria, wherein, the compounds 1-26 described herein above,are administered in combination with an additional antimalarial agentand/or an antimalarial resistance reversing agent. In general,additional antimalarial agents and/or antimalarial resistance reversingagents are ones known in the art to treat or prevent malaria, such as,for example quinolines (e.g. Chloroquine), folic acid antagonists (e.g.pyrimethamine), sulfonamides (e.g. sulfadiazine), antibiotics (e.g.tetracycline) and/or inhibitors of multidrug resistance (e.g.tetrandrine).

[0058] In general, in combination with, means, the additionalantimalarial agents and/or antimalarial resistance reversing agents maybe administered prior to, concurrent with, or subsequent to, theadministration of a therapeutically effective amount of a compoundselected from 1-26.

[0059] Compounds useful in this invention are exemplified by thefollowing examples, which should not be construed to limit the scope ofthe disclosure.

EXAMPLES Preparative Example 1

[0060]

[0061] To 2.5 kg of (R)-(−)-camphorsulfonic acid stirring at 60° C. in1250 ml of distilled water was added a solution of the potassium salt of2-carboxyl-piperazine (565 gm, 3.35 mol). The mixture was allowed tostir at 95° C. until completely dissolved. The solution was allowed tostand at ambient temperature for 48 hrs. The resulting precipitate wasfiltered to obtain 1444 gm of damp solid. The solids were then dissolvedin 1200 ml of distilled water and heated on a steam bath until allsolids dissolved. The hot solution was then set aside to cool slowly for72 hrs. The crystalline solids were filtered to give 362 gm of pureproduct (30) as a white crystalline solid. [α]_(D)=−14.9°

Preparative Example 2

[0062]

[0063] 2-R-carboxyl-piperazine-di-(R)-(−)-camphorsulfonic (362 gm, 0.608mol) was dissolved in 1.4 L of distilled water and 1.4 L of methanol. 75ml of 50% NaOH was dripped into the stirred reaction mixture to obtain a˜pH 9.5 solution. To this solution was added di-tert.butyl-dicarbonate(336 gm, 1.54 mol) as a solid. The pH dropped to ˜7.0. The pH of thereaction mixture was maintained at 9.5 with 50% NaOH (total of 175 ml),and the reaction mixture stirred for 2.5 hours to obtain a whiteprecipitate. The reaction mixture was diluted to 9 L with ice/waterfollowed by washing with 2 L of ether. The ether was discarded and thepH of the aqueous layer adjusted to pH 3.0 by the portion wise additionof solid citric acid. The acidified aqueous layer was then extractedwith dichloromethane 3× with 2L. The organic layers were combined, driedover sodium sulfate, filtered and evaporated to obtain 201.6 gm of titlecompound (31) as a white glassy solid. FABMS (M+1)=331

Preparative Example 3

[0064]

[0065] To an ice cold solution N,N-dimethylformamide (49.6 ml) wasadded, drop wise, thionylchloride (46.7 ml) over a period of 5 minutesin a 5 L round bottom flask under a nitrogen atmosphere. The reactionmixture was allowed to stir for 5 min. and the ice bath removed and thereaction mixture allowed to stir at ambient temperature for 30 min. Thereaction mixture was cooled again in an ice bath and a solution ofN,N-di-butoxycarbonyl-2-R-carboxyl-piperazine (201.6 gm, 0.61 mmol) in51.7 ml of pyridine and 1.9 L of acetonitrile was cannulated into thereaction mixture. The reaction mixture was allowed to warm to ambient toobtain a yellowish turbid solution. After stirring at ambienttemperature for 18 hours, the reaction mixture was filtered and thefiltrate poured into ice water (7L) and then extracted with 4×2 L ofethylacetate, dried over sodium sulfate, filtered and evaporated todryness under vacuo to obtain 115.6 gm (73%) of the title product (32)as a white solid.

Preparative Example 4

[0066]

[0067] Compound (32) from Preparative Example 3 (0.84 gm, 3.3 mmol) wasdissolved in 15 ml of dichloromethane. 1-(3-aminopropyl)imidazole) (0.4ml, 3.3 mmol) was added drop wise and the reaction stirred at roomtemperature for 2 hours. After 2 hours 0.2 ml more of1-(3-aminopropyl)imidazole) was added and the reaction mixture stirredfor 2 hours. Cyclohexylisocyanide (0.86 ml, 6.7 mmol) was added dropwise and the reaction mixture stirred for 4 hours. After washing withbrine, the dichloromethane was concentrated to dryness and the residuechromatographed on silica gel to obtain the title product (33) (0.9 gm)which was utilized in the next step.

Preparative Example 5

[0068]

[0069] Compound (33) from Preparative Example 4 above, was treated withtrifluoroacetic acid for 1 hour. The reaction mixture was evaporated todryness and was then evaporated from toluene 3 times to obtain theproduct (34) as an oil (0.9 gm).

Preparative Example 6

[0070]

[0071] N-Carbethoxyphthalimide (62.8 g, 0.275 mol, 1.1 eq.) was addedportion wise over a period of 30 minutes to a stirred solution ofhistamine dihydrochloride (46.7 g, 0.250 mol, 1.0 eq.) and sodiumcarbonate (54.3 g, 0.513 mol, 2.05 eq.) in distilled water (1250 ml) atroom temperature. The resulting snow-white suspension was stirredvigorously at room temperature for 90 minutes. The solid was filteredoff and thoroughly washed with ice-cold distilled water (4×50 ml). Thesolid was collected and dried under vacuum over P₂O₅ at 60° C. for 12 hto give N^(α)-phthaloylhistamine (35) (59.2 g, 0.245 mol, 98%) in highpurity (>95% by ¹H NMR). The snow-white solid (35) was used directlywithout further purification.

[0072]¹H NMR (CDCl₃, 200 MHz): δ 9.50-9.25 (br. s, 1H), 7.80-7.60 (m,5H), 6.90 (s, 1H), 3.98 (t, 2H, J=7.0 Hz), 3.06 (t, 2H, J=7.0 Hz).

[0073] MS (EI): m/e 241 (M+, 15%), 160 (34), 94 (B+).

[0074] mp 189-191° C. (ethanol, 190 proof).

[0075] Reference: Emmett, J. C., Holloway, F. H., Turner, J. L. J. Chem.Soc., Perkin Trans. 1 1979, 1341-1344.

Preparative Example 7

[0076]

[0077] A solution of chloromethyl pivalate (18.5 ml, 0.125 mol, 1.2 eq.)in anhydrous N,N-dimethylformamide (DMF, 100 ml) was added drop wiseover a period of one hour to a stirred mixture of Compound (35) (25.0 g,0.104 mol, 1.0 eq.) and potassium carbonate (17.2 g, 0.125 mol, 1.2 eq.)in anhydrous DMF (500 ml) at 90° C. under a nitrogen atmosphere. Themixture was stirred at 90° C. for 12 h. The volatiles were removed undervacuum at 50° C. The residue was taken up in brine (100 ml) andextracted with ethyl acetate (4×25 ml). The combined organic extractswere dried over Na₂SO₄, filtered, and concentrated under vacuum at 30°C. The residual off-white solid was flash-chromatographed(hexanes:acetone=6:4 v/v) over silica gel to giveN^(τ)-pivaloyloxymethyl-N^(α)-phthaloylhistamine 4 (20 g, 0.056 mol,54%) as a crystalline, white solid (36) of high purity (>95% by ¹H NMR).

[0078]¹H NMR (CDCl₃, 200 MHz): δ 7.87-7.76 (m, 2H), 7.75-7.65 (m, 3H),6.89 (s, 1H), 5.78 (s, 2H), 4.00 (t, 2H, J=7.1 Hz), 2.99 (t, 2H, J=7.1Hz), 1.14 (s, 9H).

[0079] (NOE experiments unequivocally confirmed that the N^(τ) isomerwas exclusively isolated.)

[0080] MS (FAB+): m/e 356 ([M+H]⁺).

[0081] HR-MS (FAB):Calculated for C₁₉H₂₂N₃O₄ ([M+H]⁺): 356.1610. Found:356.1613.

[0082] mp: 126-128° C.

Preparative Example 8

[0083]

[0084] A solution of Compound (36) (10.2 g, 28.7 mmol, 1.0 eq.) andα-bromo-p-tolunitrile (11.4 g, 57.4 mmol, 2.0 eq.) was stirred inanhydrous acetonitrile (150 ml) at 50° C. under a nitrogen atmospherefor 12 h. The resulting snow-white suspension was cooled to roomtemperature and chilled in a refrigerator at −20° C. for one hour. Theprecipitate was filtered off and thoroughly washed with ice-cold ethylacetate (4×50 ml). The solid was collected and dried under vacuum overP₂O₅ at 50° C. for 12 h to give Compound (37) (14.4 g, 26.2 mmol) in 91%yield. The hygroscopic salt (37) was more than 95% pure by ¹H NMR andwas used directly without any attempts at purification.

[0085]¹H NMR (CD₃OD, 200 MHz): δ 7.80-7.70 (m, 8H), 7.57-7.45 (m, 2H),6.09 (s, 2H), 5.66 (s, 2H), 3.90 (t, 2H, J=6.4 Hz), 3.05 (t, 2H, J=6.4Hz), 1.17 (s, 9H).

Preparative Example 9

[0086]

[0087] A 7 N solution of ammonia in methanol (75 ml, 0.525 mol, 7.25eq.) was added drop wise over a period of 75 minutes to a stirredsolution of Compound (37) (40 g, 0.073 mol, 1.00 eq.) in anhydrousmethanol (1000 ml) at 0° C. under a nitrogen atmosphere. The mixture wasslowly (3 h) warmed to ambient temperature and stirred for another 12 h.The volatiles were evaporated under vacuum at 30° C. and the residualwhite solid was flash-chromatographed (CH₂Cl₂:2 N NH₃/MeOH=90:10 v/v)over silica gel to give N^(τ)-(4-cyanobenzyl)-N^(α)-phthaloylhistamineCompound (38) (21 g, 0.059 mol, 81%).

[0088]¹H NMR (CDCl₃, 200 MHz): 67.87-7.70 (m, 4H), 7.68 (s, 1H), 7.63(d, 2H, J=8.4 Hz), 7.20 (d, 2H, J=8.4 Hz), 6.98 (s, 1H), 5.31 (s, 2H),3.82 (t, 2H, J=7.6 Hz), 2.81 (t, 2H, J=7.6 Hz).

[0089]¹³C NMR (CDCl₃, 300 MHz): δ 168.0, 141.4, 138.2, 134.3, 132.9,131.8, 128.2, 127.7, 127.2, 123.5, 118.3, 112.2, 48.2, 36.6, 23.0.

[0090] MS (FAB+): m/e 357 ([M+H]⁺).

[0091] Elemental Analysis: Calculated: C 70.78 H 4.53 N 15.72 Found: C70.30 H 4.52 N 15.43

Preparative Example 10

[0092]

[0093] A solution of Compound (38) (21 g, 0.059 mol, 1.0 eq.) andhydrazine monohydrate (15 ml, 0.884 mol, 15.0 eq.) in absolute ethanol(250 ml) was stirred at 50° C. under a nitrogen atmosphere for 12 h. Thesnow-white suspension was cooled to room temperature and chilled in arefrigerator at −20° C. for one hour. The precipitate (phthalylhydrazide) was filtered off and thoroughly washed with ice-cold ethanol(190 proof, 500 ml). The filtrates were combined and concentrated undervacuum at 30° C. The residue was subjected to flash columnchromatography (CH₂Cl₂:2 N NH₃/MeOH=90:10 v/v) over silica gel to giveN^(τ)-(4-cyanobenzyl) histamine (39) (11.4 g, 0.050 mol, 85%) as athick, light-brown oil.

[0094]¹H NMR (CDCl₃, 200 MHz): δ 7.64 (d, 2H, J=8.3 Hz), 7.53 (s, 1H),7.12 (d, 2H, J=8.3 Hz), 6.94 (s, 1H), 5.20 (s, 2H), 2.89 (t, 2H, J=6.8Hz), 2.54 (t, 2H, J=7.6 Hz), 1.37 (br. s, 2H).

[0095]¹³C NMR (CDCl₃, 300 MHz): 6141.9, 137.8, 132.9, 132.8, 129.6,127.7, 127.0, 118.3, 112.1, 47.9, 40.8, 28.0.

[0096] MS (FAB+): m/e 227 ([M+H]⁺).

[0097] HR-MS (FAB):

[0098] Calculated for C₁₃H₁₅N₄ ([M+H]⁺): 227.1294. Found: 227.1297.

Preparative Example 11

[0099] Step A

[0100] A solution of Compound (39) (1.50 g, 6.63 mmol, 1.0 eq.) inanhydrous dichloromethane (30 ml) was added drop wise over a period of30 minutes to a stirred solution of anhydride (32) (2.04 g, 7.95 mmol,1.2 eq.) in anhydrous dichloromethane (30 ml) at room temperature. Astream of nitrogen was bubbled through the solution to expel evolvedcarbon dioxide. The colorless solution was stirred for one hour amidnitrogen bubbling. Bubbling was terminated and cyclohexyl isocyanate(1.75 ml, 13.26 mmol, 2.0 eq.) was added drop wise over a period of 5minutes. The brown solution was stirred at room temperature for one hourto give the Boc protected piperazine intermediate (40.1) (confirmed by¹H NMR) which was reacted further without purification in Step B below.

[0101] Step B

[0102] The solution mixture containing Compound (40.1) from Step A,above, was concentrated under vacuum at 30° C. The residue was thentaken up in a mixture of trifluoroacetic acid (30 ml) and anhydrousdichloromethane (30 ml) and stirred at ambient temperature under anitrogen atmosphere for 24 h. The mixture was concentrated under vacuumat 30° C. The residual light-brown oil was taken up in 1 N aqueous NaOHsolution (100 ml) and extracted with dichloromethane (4×25 ml). Thecombined organic extracts were washed with brine (25 ml), dried overNa₂SO₄, filtered, and concentrated under vacuum at 30° C. The resultingoil was flash-chromatographed (CH₂Cl₂:2 N NH₃/MeOH=90:10 v/v) oversilica gel to give Compound (40) (1.34 g, 2.95 mmol, 45%) as alight-yellow foam.

[0103]¹H NMR (CDCl₃, 200 MHz): δ 7.97 (br. s, 1H), 7.65 (d, 2H, J=8.3Hz), 7.52 (s, 1H), 7.14 (d, 2H, J=8.3 Hz), 6.90 (s, 1H), 5.40 (d, 2H,J=6.8 Hz), 5.21 (s, 2H), 4.28 (d, 1H, J=2.6 Hz), H 3.9-1.0 (m, 21H).

[0104] MS (FAB+): m/e 464 ([M+H]⁺).

[0105] HR-MS (FAB):

[0106] Calculated for C₂₅H₃₄N₇O₂ ([M+H]⁺): 464.2774. Found: 464.2768.

Preparative Example 12

[0107] Step A

[0108] Ethyl nipecotate (70.2 g, 0.446 mmol) and D-tartaric acid (67.0g, 1.0 eq.) were dissolved in hot 95% EtOH (350 mL). The resultingsolution was cooled to room temperature, filtered, and the crystalswashed with ice-cold 95% EtOH. The product was then recrystallized from95% EtOH (550 mL) to give ethyl (S)-nipecotate D-tartrate (38.5 g, 56%yield). The salt (38.5 g) was dissolved in water (300 mL), cooled to 0°C., and 3M NaOH was added until the pH was 9-10. The resulting solutionwas extracted with CH₂Cl₂ (5×100 mL) and the combined organics driedover Na₂SO₄, filtered, and concentrated under reduced pressure to giveCompound (41) as a clear oil (19 g, 89% yield). CIMS: MH⁺=158.

[0109] Step B

[0110] To a solution of Compound (41) from Step A (18.5 g, 0.125 mol) inTHF (250 mL) was added LAH (118 mL, 1.0 M in Et₂₀, 1.0 eq.) at 0° C.over 20 minutes. The resulting solution was warmed to room temperature,then to reflux and stirred 2 hours. The reaction mixture was recooled toroom temperature and quenched by the slow addition of saturated Na₂SO₄.The resulting slurry was dried by the addition of Na₂SO₄, diluted withEtOAc (250 mL), filtered through a plug of Celite, and concentrated togive Compound (42) as a colorless oil (13.7 g 100% crude yield) whichwas used without purification. CIMS: MH⁺=116; [α]²⁰ _(D)=−8.4° (5.0 mgin 2.0 mL MeOH).

[0111] Step C

[0112] Compound (42) from Step B above (13.6, 0.104 mmol) was dissolvedin MeOH (100 mL) and H₂O (100 mL). Di-tert-butyl dicarbonate (27.2 g,1.2 eq.) was added portion wise keeping the pH>10.5 by the addition of50% NaOH. The resulting solution was stirred at room temperature for 2.5hours. The reaction mixture was concentrated in vacuo, diluted with H₂O(300 mL) and extracted with CH₂Cl₂ (3×150 mL). The combined organicswere dried over Na₂SO₄, filtered, and concentrated under reducedpressure. The crude product was purified by flash chromatography elutingwith 50% EtOH in hexane (12.1 g, 48% yield). CMS: 216 (MH⁺=100); [α]²⁰_(D)=+15.2° (5.0 mg in 2.0 mL MeOH).

[0113] Step D

[0114] pTosCl (12.8 g, 1.2 eq.) was added portion wise to Compound (43)from Step C (12.0 g, 55.7 mmol) in pyridine (120 mL) at 0° C. Theresulting solution was stirred 0° C. overnight. The reaction mixture wasdiluted with EtOAc (300 mL) and washed with cold 3N HCl (5×300 mL),saturated NaHCO₃ (2×150 mL), H₂O (1×100 mL), brine (1×100 mL), driedover Na₂SO₄, filtered, and concentrated in vacuo to give Compound (44)as a pale yellow oil which was used without purification (100% crudeyield). FABMS: MH⁺=370.

[0115] Step E

[0116] Compound (44) from Step D above (21.0 g, 5.7 mmol) in DMF (300mL) was treated with sodium imidazole (8.37 g, 1.5 eq.). The resultingsolution was stirred, heated to 60° C., and stirred 2 hours. Thereaction mixture was cooled to room temperature and concentrated invacuo. The residue was diluted with H₂O (300 mL) and extracted withCH₂Cl₂ (3×150 mL). The combined organics were dried over Na₂SO₄,filtered, and concentrated under reduced pressure. The crude product waspurified by flash chromatography using a 7% MeOH in CH₂Cl₂ solution aseluent to give Compound (45) as a pale yellow solid (7.25 g, 49% yield).FABMS: MH⁺=266; [α]²⁰ _(D)=+8.00 (5.0 mg in 2.0 mL MeOH).

[0117] Step F

[0118] The title compound (45) from Step E (5.50 g, 20.7 mmol) wasstirred at room temperature in 4M HCl/dioxane (50 mL) overnight. Theresulting solution was concentrated in vacuo and triturated with Et₂O togive Compound (46) as a yellow solid. CIMS: MH⁺=166.

Preparative Example 13

[0119]

[0120] 2-Carboxy-piperazine-dicamphorsulfonic acid salt (30)(PreparativeExample 1) (17.85 gm, 30 mmole) was dissolved in 180 ml of distilledwater. Dioxane (180 mL) was added and the pH adjusted to 11.0 with 50%NaOH. The reaction mixture was cooled to 0-5° C. in an ice-MeOH bath anda solution of benzyl-chloroformate (4.28 mL, 30 mmol) in 80 mL ofdioxane was added over a period of 3045 minutes while stirring at 0-5°C. and keeping the pH at 10.5 to 11.0 with 50% NaOH. After the additionwas complete, stirring was continued for 1 hr. The reaction mixture wasthen evaporated to dryness (to get rid of the dioxane for extraction).The residue was dissolved in 180 mL of distilled water and the pHadjusted slowly to 4.0 with 1N HCl. The aqueous solution was washed with3×180 mL of ethyl acetate (The ethyl acetate was dried over MgSO₄,filtered, and evaporated to obtain N,N-di-CBZ-2-carboxy-piperazine andsaved). The pH of the aqueous layer, which contains the desired product,was adjusted to 10.5 to 11.0 with 50% NaOH and soliddi-tert-butyl-dicarbonate (7.86 gm, 36 mmol) was added and the mixturewas stirred while keeping the pH at 10.5 to 11.0 with 50% NaOH. After 1hr. the pH stabilized. When the reaction was complete, the reactionmixture was washed with 2×1 80 mL of Et₂O. The aqueous layer was cooledin an ice bath and the pH was adjusted to 2.0 with 1N HCl (slowly). Thereaction mixture was extracted with 3×200 mL of ethyl acetate, driedover MgSO₄, filtered and concentrated to obtain 9.68 gm (88%) of pureproduct (47) as a white solid.

Preparative Example 14

[0121]

[0122] Compound (47) from Preparative Example 13 (9.6 gm, 26.3 mmol) wasdissolved in 100 mL of absolute ethanol in a hydrogenation vessel. Thevessel was flushed with nitrogen and 3 gm of 10% Pd/C (50% by weightwith water) was added. The mixture was hydrogenated at 55 psi of H₂ for18 hours. After 18 hrs, the reaction mixture had a precipitate. The TLCwas checked (30% MeOH/NH₃/CH₂Cl₂). The reaction mixture was filtered ona pad of Celite, and the pad washed with EtOH followed by distilledwater. The filtrate was evaporated to ˜⅓ the volume (to get rid of theEtOH) and 200 mL of distilled water was added. The aqueous layer wasextracted with ethyl acetate three times (the ethyl acetate layercontaining pure N,N-Di-Boc-2-carboxy-piperazine was saved). The waterlayer was evaporated to dryness and evaporated from methanol two timesto obtain 3.98 gm of pure product (48).

Preparative Example 15

[0123] Step A

[0124] The tricyclic alcohol (Preparative Example 40 in WO 95/10516)(5.6gm, 17.33 mmol) was dissolved in 56 ml of dichloromethane and 2.46 ml ofthionyl chloride was added while stirring under a dry nitrogenatmosphere. After 5 hours the TLC was checked (by adding an aliquot ofthe reaction mixture to 1N NaOH and shaking with dichloromethane andchecking the dichloromethane layer by TLC using 50% EtOAc/Hexanes as theeluent). The mixture was evaporated to give a gum, which was evaporated,twice from dry toluene and once from dichloro-methane to give the11-chloro derivative (49) as a foamy solid, which was used withoutfurther purification.

[0125] Step B

[0126] Compound (49) from Step A above, was dissolved in 100 ml of dryDMF, and Compound (48) from Preparative Example 14 (3.98 gm) was addedfollowed by 12.11 ml of triethylamine. The mixture was stirred atambient temperature under a nitrogen atmosphere. After 24 hours the DMFwas evaporated and the residue dissolved in 200 ml of ethyl acetate andwashed with brine. The brine layer was washed with ethyl acetate twomore times and the ethyl acetate layers combined, dried over magnesiumsulfate, filtered, and evaporated to give a foamy solid. The solid waschromatographed on a 1½″×14″ column of silica gel eluting with 2L of0.4% 7N MeOH/NH₃:CH₂Cl₂, 6L of 0.5% 7N MeOH/—NH₃:CH₂Cl₂, 2L of 0.65% 7NMeOH/NH₃:CH₂Cl₂, 2L of 0.8% 7N MeOH/NH₃:CH₂Cl₂, 4L of 1% 7NMeOH/NH₃:CH₂Cl₂, 2L of 3% 2N MeOH/NH₃:CH₂Cl₂, 2L of 5% 2NMeOH/NH₃:CH₂Cl₂, 2L of 10% 2N MeOH/NH₃:CH₂Cl₂, 2L of 15% 2NMeOH/NH₃:CH₂Cl₂, 4L of 20% 2N MeOH/NH₃:CH₂Cl₂ to obtain 4.63 gm of finalproduct (50).

Preparative Example 16

[0127]

[0128] A mixture of 1-(3-aminopropyl)imidazole (37.1 g, 297 mmol),benzaldehyde (30 g, 283 mmol), 3 Å molecular sieves (50 g), sodiumacetate (24.1 g, 283 mmol) and anhydrous methanol (700 mL) was stirredat room temperature under N₂ overnight. The mixture was cooled to 0° C.and sodium borohydride (10.9 g, 288 mmol) was added portion wise over 1hour. The mixture was stirred at room temperature for 3 hours. Themixture was filtered through celite, washed with methanol, andconcentrated in vacuo to give a residue, which was diluted withdichloromethane and washed with 10% aqueous sodium hydroxide. Theorganic phases were washed with brine, dried over anhydrous magnesiumsulfate, filtered and concentrated in vacuo to give the title compoundas a pale yellow oil (56.3 g, 92%, MH⁺=216).

Preparative Example 17

[0129]

[0130] In essentially the same manner as Preparative Examples 4 and 5,except substituting methanesulfonyl chloride for cyclohexylisocyanide,Compound (52) was prepared.

Preparative Example 18

[0131]

[0132] In essentially the same manner as Preparative Examples 4 and 5,except substituting 1-(2-aminoethyl)imidazole) for1-(3-aminopropyl)imidazole), Compound (53) was prepared.

Preparative Example 19

[0133]

[0134] Compound (32) from Preparative Example 3 was treated with1-(3-aminopropyl)imidazole in essentially the same manner as PreparativeExample 4 to afford the t-Boc derivative, which was further reacted in asimilar manner as in Preparative Example 5 to afford Compound (54).

Preparative Example 20

[0135] Step A

[0136] To a solution of 1,2-iodobromobenzene (11.5 mL) in 840 ml oftetrahydrofuran, cooled to −78° C. and under a dry nitrogen atmospherewas added 45.5 mL of a 2M ether solution of isopropylmagnesiumchloride.After stirring for 30 minutes, a solution of3-bromo-5,6-dihydro-2H-pyrano[3,4-b]pyridin-8-one (14 gm, 61.67 mmol) in100 mL of tetrahydrofuran was added and the reaction mixture stirred for1 hr. The reaction was then poured into a solution of saturated ammoniumchloride and the resulting mixture extracted three times with ethylacetate. The combined ethyl acetate layers were then dried overmagnesium sulfate, filtered, and evaporated to give a solid. The solidwas crystallized from ethylacetate/hexanes to obtain 21.82 g of compound(55) FABMS M+1=384.

[0137] Step B

[0138] Compound (55) from Step A (15 gm, 39 mmol) was mixed with 67 gmof aluminum chloride and the mixture was heated to 160° C. in a roundbottom flask for 2 hours. While cooling the reaction in an ice bath,ice, followed by 500 ml of water was added. Sodium hydroxide (50%) wasadded to adjust the pH to 12. The resulting solution was extracted threetimes with ethyl acetate to obtain 9.75 gm of a crude mixture ofproducts which was further purified by chromatography to obtain 1.25 gmof compound (56) FABMS M⁺1=365.

[0139] Step C

[0140] To a solution of Compound (56) from Step B (1.2 gm, 3.3 mmol) in20 ml of methanol at room temperature was added, portion wise, sodiumborohydride (0.29 gm, 5 mmol). After 1 hour, 30 ml of 1N hydrochloricacid was added and the mixture stirred for 5 minutes. To the reactionwas added, 1N sodium hydroxide (50 ml) and the resulting mixtureextracted with dichloromethane to obtain 1.18 gm of title compound whichwas used directly in the next step without further purification FABMSM⁺1=367.

[0141] Step D

[0142] Compound (58) from Step C, was treated in a manner similar tothat described in Preparative Example 15, Step A, to obtain compound(59)

Preparative Example 21

[0143] Step A Preparation of Compound (60).

[0144] Loratadine® (448 g, 1.17 mol) was refluxed in 2 L of 70% aqueousHCl (1.4 L conc.HCl in 600 ml H₂O) for 12 h. The reaction mixture wasthen cooled and poured into ice. It was then basified with 950 mL of 50%NaOH followed by extraction with CH₂Cl₂ (1×4L, and 2×2.5L). The organicphase was washed with brine, dried over Na₂SO₄ and MgSO₄ and thenfiltered. All the volatiles were then removed to give 368 g of the titlecompound (60). MH⁺=311

[0145] Step B Preparation of Compound (61).

[0146] To the title compound from Preparative Example 21, Step A (363 g,1.17 mol) was added trifuromethane sulfonic acid (1.8 Kg) under N₂. Thereaction mixture was refluxed at 170° C. The progress of the reactionwas monitored by ¹H NMR. After 4 days the reaction was only 63%complete. After 8 days the reaction was found to be 80% completeaccording to ¹H NMR; thus another 130 mL of CF₃SO₃H was added andrefluxing continued for another 24 h. It was then poured into ice andbasified with 800 mL of NaOH (50%) and extracted twice with CH₂Cl₂ (1×8Lthen 1×7L). The organic phase was combined, washed with H₂O and filteredthrough celite. It was then dried over MgSO₄ and Na₂SO₄ and againfiltered through celite. The filtrate was concentrated to give a blackbrown semi-solid that was pre-adsorbed on 600 g of silica gel and thenchromatographed on 2.3 Kg of silica gel eluting first with 5%CH₃OH—CH₂Cl₂ (saturated with ammonia) and then with 10% CH₃OH—CH₂Cl₂(saturated with ammonia) to give 102 g of the title compound (61) as asolid. mp=73-75; MS (FAB) m/z 483 (MH⁺).

[0147] Step C Preparation of Compound (62).

[0148] To a solution of the title compound of Preparative Example 21,Step B (145 g) in 1L of CH₂Cl₂ at 0° C. was added ethylchloroformate (55mL), dropwise. The reaction mixture was stirred at room temperatureovernight. It was further diluted with 1L CH₂Cl₂ and stirred with 2L ofdilute NaHCO₃, pH ˜7-8. The organic layer was separated and dried overMgSO₄ and Na₂SO₄, filtered and concentrated to afford 174 g of a brownblack gum. The crude compound was purified by silica gel columnchromatography, eluting with 20-60% ethyl acetate-hexane to afford thetitle compound (62). MS (FAB) m/z 383 (MH⁺).

[0149] Step D Preparation of compounds (63) and (64).

[0150] The title compound of Preparative Example 21, Step C (251 g, 0.65mol) was dissolved in 1.65 L of CH₂Cl₂ and dibromo-dimethylhydantoin(132 g, 0.462 mol) was then added. The solution was stirred until thesystem was homogeneous. The solution was cooled to 0° C. under N₂atmosphere and 174 mL of CF₃SO₃H was added over 37 min. while keepingtemperatures between ⁻1° C. to ⁺1° C. The reaction mixture was stirredfor 3 h, cooled to ⁻10° C. and basified with 50% NaOH (170 mL), keepingthe temperature below ⁺1° C. The aqueous phase was extracted with CH₂Cl₂and then dried over MgSO₄ and concentrated to give 354 g of yellow foamthat was chromatographed on silica gel eluting with 10-50% of ethylacetate-hexanes gradient to give 50 g of compound (63) (14% yield) and147 grams of the desired title compound (64) (49% yield). Compound (64)MS m/z (rel intens) 462 (MH⁺); Compound (63) MS m/z (rel intens) 542(MH⁺).

[0151] Step E Preparation of Compound (65)

[0152] Compound 64 from Preparative example 21, Step D (10.0 g, 21.7mmol) was refluxed in 2 L of 70% aqueous HCl (1.4 L conc. HCl in 600 mlH₂O) for 12 h. The reaction mixture was then cooled and poured into ice.It was then basified with 950 mL of 50% NaOH followed by extraction withCH₂Cl₂ (1×4L, and 2×2.5L). The organic phase was washed with brine,dried over Na₂SO₄ and MgSO₄, and then filtered. All the volatiles werethen removed to give the title compound (65). MH⁺=389.

[0153] Step F Preparation of Compound (66)

[0154] To a solution of the amine product (65) from Preparative Example21, Step E (20.0 g) in CH₂Cl₂ (100 ml) was added triethyl amine (14.4ml). Slowly, methane sulfonyl chloride (6.0 ml) was added and themixture stirred over night at room temperature. To the reaction wasadded saturated sodium bicarbonate and then it was extracted withCH₂Cl₂. The organic layer was dried over magnesium sulfate, filtered andconcentrated to dryness. The crude product mixture was purified withcolumn chromatagraphy, eluting with 1% MeOH/NH₃—CH₂Cl₂, to afford thedesired compound (66). MS 469 (MH⁺).

[0155] Step G Preparation of Compound (67) and (68)

[0156] To the title compound from Preparative Example 21, Step F (21.25g, 45.3 mmol, 1 eq.) in 400 mL of THF was added 19.5 g (266.6 mmol, 5eq.) of piperazine. The reactants stirred at room temperature untileverything was in solution. To this mixture was added potassiumt-butoxide 12.7 g (113.3 mmol, 2.5 eq.) in one portion. The reactionmixture was stirred at room temperature for 2 h. All of the THF wasremoved by rotary evaporation and the resulting crude product wasextracted with EtoAc and washed with water. The organic layer was driedover Na₂SO₄ and concentrated to give a mixture of title compounds (67)and (68).

[0157] Step H Preparation of Compound (69) and (70)

[0158] The product from Preparative Example 21, Step G (22.25 g) wasdissolved in 150 mL of conc. HCl and stirred for 16 h. The reactionmixture was poured into ice, basified with conc. NH₄OH and thenextracted with CH₂Cl₂ to give a mixture of compounds (69) and (70).

Preparative Example 22

[0159] Step A Compounds (71 & 72)

[0160] To a mixture of piperazinyl compounds 69 & 70, from PreparativeExample 21, Step H in THF (150 mL) at −78° C. was added LDA 4.05 mL (1.1eq.) and the solution stirred for 1.5 h. The mixture was warmed to −20°C. and then N-phenyl trifluoromethane sulfonimide 2.94 g (1.1 eq.) wasadded. The reaction stirred over night at room temperature. After all ofthe THF was removed by rotatary evaporation, the resulting crude productwas purified by Biotage column chromatography eluting with 50%EtoAc-Hex, (normal phase) to yield 1.94 g of the desired compound (71).

[0161] Step B Preparation of Compound (73)

[0162] Compound (71) from Preparative example 22, Step A above, wasdissolved in DMF. Successively, Et₃N (29 eq.), Ethyl acrylate (5.4 eq.),K₂CO₃ (5 eq.), Bu₄NBr (2 eq.) and Palladuim (II) acetate (0.13 eq.) wereadded. The mixture was stirred and heated to 100° C. for 4 h. Aftercooling, the mixture was concentrated and the residue was taken up inCH₂Cl₂ and extracted with CH₂Cl₂/H₂O. The organic layer was dried overNa₂SO₄ then concentrated and the residue purified by Biotagechromatography using 1% MeOH/NH₃—CH₂Cl₂ to afford the title compound(73).

[0163] Step C Preparation of Compound (74)

[0164] Compound (73) was dissolved in EtOH, cooled in an ice bath andreacted with NaBH₄ (15 eq.) for 3 min. Then CuCl (2 eq) was added andthe reaction mixture was stirred for 6 h. at room temperature. Themixture was filtered, concentrated and extracted with CH₂Cl₂. Theorganic layer was washed with sat. NaHCO₃, dried over Na₂SO₄ andconcentrated to yield the title compound (74).

[0165] Step D Preparation of Compound 75

[0166] To a solution of Compound 74 (1.24 g) in CH₂Cl₂ (100 ml) wasadded triethyl amine (1.1 ml). Slowly, methane sulfonyl chloride (0.3ml) was added and the mixture stirred over night at room temperature. Tothe reaction was added saturated sodium bicarbonate and then it wasextracted with CH₂Cl₂. The organic layer was dried over magnesiumsulfate, filtered and concentrated to dryness. The crude product mixturewas purified with column chromatography, eluting with 80% EtOAc-Hex, toafford the title compound (75).

Preparative Example 23

[0167] Step A Preparation of Compound 78

[0168] To a stirred solution of bromine (33.0 g, 210 mmol) in CCl₄ (100ml) was added a solution of dibenzosuberenone (37.0 g, 179 mmol) in CCl₄(200 ml) at room temperature. The resulting solution was stirred at roomtemperature for 1.5 hours. The white crystals were collected byfiltration to give the product (78) (60.12 g, 92% yield, M+H=367).

[0169] Step B Preparation of Compound (79)

[0170] A solution of the di-bromo compound (78) (10.0 g, 27.3 mmol) andNaOH (3.0 g, 82.0 mmol) in MeOH (200 ml) was stirred and heated toreflux for 1.5 hours. The reaction mixture was then cooled to roomtemperature and stirred overnight. The mixture was evaporated to drynessand then extracted with CH₂Cl₂—H₂O. The combined organic layer was driedover MgSO₄, filtered and evaporated to dryness to give a white solid(79) (8.0 g, 100% yield, M=285)

[0171] Step C Preparation of Compound 80

[0172] To a stirred solution of the mono-bromo compound (79) from step B(3.9 g, 13.7 mmol) in MeOH (200 ml) under nitrogen and at 0° C. wasadded NaBH₄ (0.7552 g, 20.0 mmol). The resulting solution was stirred at0° C. for 3 hours, then evaporated, followed by extraction withCH₂Cl₂—H₂O. The combined organic layer was dried over MgSO₄, filtered,and evaporated to dryness to give a white solid (80) (4.1 g, 100%,M=287).

[0173] Step D Preparation of Compound 81

[0174] To a stirred solution of alcohol (80) from Preparative Example 3,Step C (3.9 g, 13.6 mmol) in CH₂Cl₂ (100 mL) under nitrogen at −20° C.was added thionyl chloride (13.9 mL, 1.0 mmol). The resulting solutionwas stirred at room temperature overnight and then evaporated todryness. The crude mixture was diluted with toluene (50 mL), followed bythe addition of more SOCl₂ (13.9 mL) at room temperature. The resultingsolution was heated to reflux for 2 hours until the reaction went tocompletion. The reaction mixture was then cooled to room temperature andconcentrated to dryness to give a light brown solid (81) (5.0 g, 100%yield, M-BrCl=191)

[0175] Step E Preparation of Compound 82

[0176] To a suspension of Mg (3.63 g) in anhydrous THF (95 mL) undernitrogen and at room temperature was added 4-chloro-1-methyl piperidine(3 mL, 10% of the total amount) and one small crystal of iodine. Theresulting solution was heated to reflux, followed by the addition ofiodomethane (0.5 mL) and the remainder of the 4-chloro-1-methylpiperidine (27 mL). The reaction was stirred for one hour and thenconcentrated to dryness to give the crude Grignard reagent (0.8M).

[0177] To a stirred solution of the chloro compound (81) fromPreparative Example 3, Step D (3.9 g, 13.7 mmol) in anhydrous THF (40mL) under nitrogen at 0° C. was added dropwise the Grignard reagent(obtained above) (0.8M, 19.5 mL, 15.6 mmol). The resulting solution wasstirred at 0° C. for 1 hour. The reaction mixture was quenched with 100mL of 15% aq. NH₄Cl solution, followed by the extraction with EtOAc-H₂O.The combined organic layer was dried over MgSO₄, filtered and evaporatedto dryness to give the product (82) (5.29 g, 100% yield, MH⁺=368).

[0178] Step F Preparation of Compound 83

[0179] To a stirred solution of Compound (82) from Step E above (5.0 g,13.6 mmol) in toluene (100 mL) under a nitrogen atmosphere, was addedtriethylamine (5.7 mL, 40.7 mmol). The resulting solution was heated torefux, then dropwise ethyl chloroformate (13.0 mL, 136.7 mmol) wasadded. The solution continued to stir at the reflux temperature for 2hours. The reaction was then stirred at room temperature overnight,followed by extraction with an EtOAc-1N NaOH solution. The combinedorganic layer was dried over MgSO₄, filtered, concentrated to drynessand the crude product purified by column chromatography on normal phasesilica gel, eluting with 10% EtOAc/90% Hexane to give (83).

[0180] Step G Preparation of Compound 84

[0181] The product from Preparative Example 23, Step E was dissolved in25 mL of conc. HCl and heated to reflux overnight. The reaction mixturewas poured into ice basified with 50% w/w NaOH and then extracted withCH₂Cl₂ to give the desired compound 84.

[0182] Step H Preparation of Compound 85

[0183] To a solution of the amine (84) (0.82 g) in CH₂Cl₂ (10 ml) wasadded triethyl amine (1.0 ml). Slowly, methane sulfonyl chloride (0.3ml) was added and the mixture stirred over night at room temperature. Tothe reaction was added saturated sodium bicarbonate and then it wasextracted with CH₂Cl₂. The organic layer was dried over magnesiumsulfate, filtered and concentrated to dryness to give the desiredcompound (85).

[0184] Step I Preparation of Compound 86

[0185] Compound (85) from Preparative Example 23 Step H, was dissolvedin DMF. Successively, Et₃N (29 eq.), Ethyl acrylate (5.4 eq.), K₂CO₃ (5eq.), Bu₄NBr (2 eq.) and Palladuim (II) acetate (0.13 eq.) were added.The mixture was stirred and heated to 100° C. for 4 h. After cooling,the mixture was concentrated and the residue was taken up in CH₂Cl₂ andextracted with CH₂Cl₂/H₂O. The organic layer was dried over Na₂SO₄ thenconcentrated and the residue purified by normal phase silica gel using25% EtOAc-75% hexane to afford the title compound (86).

[0186] Step J Preparation of Compound 87

[0187] Compound (86) from Step I above was dissolved in EtOH, followedby the addition of CH₂Cl₂ at room temperature. To this reaction solutionwas added 10% w/w of PtO₂ and the reaction stirred at room temperatureunder H₂ (balloon) overnight. The reaction mixture was filtered throughcelite and concentrated to yield the title compound 87.

[0188] Step K Preparation of Compound 88

[0189] To a stirred solution of the ester (87) from Step J above, (0.63g, 1.4 mmol) in THF (10 mL) at 0° C. was added a 1M solution of DIBAL(2.8 mL, 2.8 mmol). The resulting solution was stirred at roomtemperature overnight. An additional portion of 1M DIBAL was added andthe mixture was stirred for 4 more hours at 0° C. The reaction solutionwas extracted with EtOAc-10% citric acid, 1N NaOH. The combined organiclayer was dried over Na₂SO₄ filtered and evaporated to give titlecompound 88.

[0190] Step L. Preparation of Compound 89.

[0191] In a similar manner as was described in Preparative example 22,Step D, triethyl amine was added to a solution of Compound (88) inCH₂Cl₂ (100 ml). Slowly, methane sulfonyl chloride was added and themixture stirred over night at room temperature. To the reaction wasadded saturated sodium bicarbonate and then it was extracted withCH₂Cl₂. The organic layer was dried over magnesium sulfate, filtered andconcentrated to dryness. The crude product mixture was purified withcolumn chromatography, eluting with 80% EtOAc-Hex, to afford the titlecompound (89).

Preparative Example 24

[0192] Step A Preparation of Compound (90).

[0193] The tricyclic keto-compound (disclosed in U.S. Pat. No.5,151,423) (30.0 g; 123.2 mmol) was combined with NBS (48.2 g; 271.0mmol) and benzoyl peroxide (0.42 g) in CCl₄ (210 ml). The reaction washeated to 80° C. for 10 hr. The mixture was cooled and let stand for 8hr. The resulting precipitate was filtered, added to MeOH (200 ml) andthe resulting mixture was stirred over 2 days. The solid was filteredand dried under vacuum to a constant weight.

[0194] Step B Preparation of Compounds (91a) and (91b)

[0195] The dibromo compound (90) from Step A (35.72 g; 88.97 mmol) abovewas dissolved in CH₂Cl₂ (1.5 L) and cooled to 0° C. Dropwise, DBU (15.96ml) was added and the suspension stirred for 3 hr. The reaction mixturewas concentrated redissolved in CH₂Cl₂ (1.5 L) filtered through a bed ofsilica gel and rinsed with 5% EtOAc/CH₂Cl₂ (4 L). The combined rinseswere concentrated and purified by flash silica gel column chromatographyinto pure 5 and 6 mono-bromo substituted compounds eluting with 10-30%EtOAc/Hex, then 3% EtOAc/CH₂Cl₂.

[0196] Step C Preparation of Compound (92).

[0197] The 5-bromo substituted compound (91a) from Step B above (4.0 g;12.45 mmol) was taken up in MeOH and cooled to 0° C. NaBH₄ (916.4 mg;24.2 mmol) was added and the reaction mixture stirred for 5.5 hr. Thesolvent was removed and the resulting residue was used directly.

[0198] Step D Preparation of Compound (931.

[0199] The alcohol compound (92) from Step C above (3.98 g; 12 mmol) wasdissolved in CH₂Cl₂ cooled to 0° C. and treated with 2,6-Lutidine (5.73ml; 49 mmol). SOCl₂ (1.8 ml; 24.6 mmol) was added and the reaction wasallowed to stir and come to room temperature over 3 hr. The reactionmixture was poured into 0.5 N NaOH (80 ml) extracted and concentrated invacuo. The crude product was taken up in CH₃CN and treated with1,2,2,6,6-Pentamethylpiperidine (4.45 ml; 24.6 mmol) (Aldrich). Thereaction was heated to 60-65° C. treated with tert-butyl1-piperazinecarboxylate (2.32 g; 12 mmol) (Aldrich) and stirred overnight under N₂ atmosphere. The reaction mixture was concentrated todryness, redissolved in CH₂Cl₂ and washed with sat. aqueous NaCO₃. Theorganic layer was dried over Na₂SO₄, filtered and purified by flashsilica gel column chromatography eluting with 1:4-1:2 EtOAc/Hexanes toafford the product as a white solid.

Preparative Example 25 Compound (94).

[0200]

[0201] In essentially the same manner as in Preparative Example 24,Steps C-D, substituting the 6-Bromo substituted compound (91b) from StepB, for the 5-Bromo substituted compound (91a), Compound (94) wasprepared (76.6 g, 100% yield).

Preparative Example 26

[0202] Step A Preparation of Compound (95).

[0203] To a solution of Compound (94) from Preparative Example 25 (4.0g, 8.16 mmol) in toluene (75 mL) and MeOH (20 mL), was added triphenylphosphine (1.099 g, 4.08 mmol), DBU (1.7 g, 11.02 mmol) and palladiumchloride (0.145 g, 0.82 mmol). The resulting solution was evacuated withCO at 100 psi and heated at 78° C.-82° C. for 5 hours, followed by theextraction with EtOAc-H₂O. The combined organic layer was then washedwith brine, dried over Na₂SO₄, concentrated to dryness and purified bycolumn chromatography, eluting with 30% EtOAc/70% Hexane to giveCompound (95) (3.12 g, 100% yield, MH⁺=470.1).

[0204] B. Preparation of Compound (96).

[0205] A solution of Compound (95) from Step A above (3.1 g, 6.6 mmol)in 4M HCl/Dioxane (120 mL) was stirred for 3 hours and then concentratedto dryness to give the crude salt of Compound (96) (3.89 g, 100% yield,MH⁺=370.2)

[0206] Step C Preparation of Compound (97).

[0207] To a solution of Compound (96) from Step B above (3.43 g, 8.45mmol) in THF (60 mL) at 0° C., was added DIBAL (7.21 g, 50.7 mmol). Theresulting solution was warmed to room temperature, stirred overnight andthen concentrated to dryness, followed by the addition of Boc anhydride(3.69 g, 16.9 mmol). The reaction was then extracted with CH₂Cl₂—H₂O,filtered over Na₂SO₄ and concentrated to dryness to afford Compound (97)(3.75 g, 100% yield, MH⁺=442.4).

[0208] Step C.1 Alternate Preparation of Compound (97).

[0209] A solution of compound 95 from Step A above (23.46 g, 50.98 mmol)in CH₂Cl₂—MeOH—H₂O (120 mL, 600 mL, 60 mL respectively) combined withLiOH (12.0 g, 350.88 mmol) was refluxed at 40° C. overnight. Solvent wasremoved from the reaction mixture. The residue was diluted with CH₂Cl₂then acidified to pH 6 with 1N HCl. The organic layer was separated andwashed with water, dried over Na₂SO₄ and concentrated. The product wasdissolved in THF (285 mL) at 0° C. Triethyl amine (6 mL, 42.97 mmol) andethyl chloroformate (4.1 mL, 42.97 mmol) were added and stirred at 0° C.for 1 h. The reaction mixture was filtered and the filtrate was cooledto −70° C. To this filtrate was added NaBH₄ (3.97 g, 104.94 mmol) andstirred for 1 h at −70° C. after which time 40 mL of MeOH was addeddropwise. The solvents were removed and the residue taken up inmethylene chloride, washed with sat. (aq) NaHCO₃, then brine, dried overNa₂SO₄ and concentrated to give Compound (97) as a solid.

[0210] Step D Preparation of Compound (98).

[0211] To a solution of Compound (97) from Step C above (3.74 g, 8.46mmol) in CH₂Cl₂ (100 mL) was added triethyl amine (3.5 mL, 25.38 mmol)and methanesulfonyl chloride (1.45 g, 2.7 mmol). The resulting solutionwas stirred under nitrogen at room temperature for overnight and thenwashed with saturated NaHCO₃, then brine, and dried over Na₂SO₄ to givethe mesylate compound (98) (3.86 g, 88% yield).

[0212] Step E Preparation of Compounds (99a) and (99b)

[0213] To a solution of 2-methylimidazole (2.43 g, 29.68 mmol) in DMF(30 mL) under N₂ was added NaH (0.53 g, 22.3 mmol) and stirred for 10min, followed by the addition of Compound (98) from Step D above (3.86g, 7.42 mmol). The solution was stirred over night. The solution wasthen concentrated to dryness and extracted with EtOAc-NaHCO₃, dried overNa₂SO₄, and concentrated. The crude product was purified by columnchromatography, eluting with 2% MeOH—NH₃/98% CH₂Cl₂ to afford a mixtureof isomers. Further separation was accomplished by Preparative HPLCChiral AD Column chromatography, eluting with 25% IPA/75% hexane/0.2%DEA to give pure Compound (99a) (isomer 1) (0.160 g) and Compound (99b)(isomer 2) (0.140 g) (MH⁺=506.1)

[0214] Step F Preparation of Compounds (100a) and (100b).

[0215] A solution of Compound (99a) (isomer 1) from Step E above (0.105g, 0.21 mmol) in 4M HCl/Dioxane (10 mL) was stirred at room temperaturefor 3 hours and concentrated to dryness to afford Compound (100a) (0.147g, 100% yield)

[0216] Compound (99b) (isomer 2) from Step E was treated in the samemanner as isomer 1 above, to afford Compound (100b) (isomer 2).

Examples 1 and 2

[0217]

[0218] Sodium triacetoxyborohydride (75 mg, 0.336 mmol, 3.1 eq.) wasadded portion wise (3×25 mg) to a stirred solution of compound (40) (50mg, 0.108 mmol, 1.0 eq.) from Preparative Example 11, Step B, and theappropriate aldehyde of the R group shown in the table below (0.336mmol, 3.1 eq.), in a mixture of glacial acetic acid (0.5 ml) andanhydrous dichloromethane (10 ml) at 0° C. under a nitrogen atmosphere.The mixture was slowly (3 h) warmed to room temperature and stirred foranother 12 h. The volatiles were removed under vacuum at 30° C. Theresidue was taken up in 1N aqueous NaOH solution (10 ml) and extractedwith dichloromethane (5×5 ml). The combined organic extracts were washedwith brine (5 ml), dried over Na₂SO₄, filtered, and concentrated undervacuum at room temperature. The product was purified by preparativescale thin layer chromatography (using either CH₂Cl₂:2 N NH₃/MeOH=90:10v/v or CH₃CN:2 N NH₃/MeOH=90:10 v/v as eluent) over silica gel to affordthe compounds listed in the Table A below:

[0219] Reference: Abdel-Magid, A. F., Maryanoff, C. A., Carson, K. G.Tetrahedron Lett. 1990, 31, 5595. TABLE A Example # R Compound #Physical Data 1

21 MS: MH+ = 611.3 mp 100° C. (dec) 48% yield 2

22 MS: MH+ = 555.3 mp 80° C. (dec) 38% yield

Example 3

[0220]

[0221] TEA (0.66 mL, 5.0 eq.) was added drop wise to a stirred solutionof Compound (60) (0.25 g, 0.946 mmol) (see U.S. Pat. No. 5,151,423) andCompound (46) from Preparative Example 12, Step F (0.25 g, 1.1 eq.) inCH₂Cl₂ (5.0 mL). The resulting solution was stirred at room temperaturefor 24 h. then diluted with H₂O, neutralized with saturated NaHCO₃, andextracted with CH₂Cl₂. The combined organics were dried over Na₂SO₄,filtered, and concentrated under reduced pressure. The crude product waspurified by flash chromatography using a 5% (10% NH₄OH in MeOH) inCH₂Cl₂ solution as eluent to yield Compound (20) as the first elutingisomer and a mixture of diastereomers:

[0222] Compound (20):LCMS: MH⁺=393; mp=71-75° C.; [α]_(D) ²⁰=−65° (3.97mg in 5.0 mL MeOH).

Example 4

[0223]

[0224] Compound (49) from Preparative Example 15, Step A, was reactedwith Compound (52) from Preparative Example 17 using essentially thesame procedure as in Example 3, to obtain Compound (23); FABMS(M+1)=623.

Example 5

[0225]

[0226] Compound (34) (0.9 gm) from Preparative Example 5 was reactedwith compound (59) from Preparative Example 20, Step D 0.36 gm, 1 mmol)using essentially the same procedure as in Example 3, to obtain Compound(16); FABMS (M+1)=715.

Example 6

[0227]

[0228] Compound (53) from Preparative Example 18 was reacted withCompound (49) from Preparative Example 15, Step A, using essentially thesame procedure as in Example 3, to obtain Compound (17); FABMS(M+1)=656.

Example 7 Compound 18

[0229]

[0230] Compound (54)(0.9 gm) from Preparative Example 19 was reactedwith Compound (49) from Preparative Example 15, Step A, usingessentially the same procedure as in Example 3, to obtain Compound (18).

Example 8 Compound 19

[0231]

[0232] Compound (16) from Example 5 was further purified by HPLC on aChiralcel AD column eluting with 30% Isopropanol/Hexanes: 0.2% DEA, toafford the pure isomer A Compound (19).

Example 9 Compounds 13 and 13a

[0233]

[0234] To a solution of compound (75) from Preparative example 22, StepD (0.2 g, 0.41 mmole) in DMF (7 ml) was added imidazolylsodium (0.054 g,0.61 mmol). The reaction mixture was heated to 90° C. for 2 h thencooled and the DMF was removed. To the residue was added saturatedsodium bicarbonate and the solution was extracted with CH₂Cl₂. Theorganic layer was dried over magnesium sulfate, filtered andconcentrated to dryness. The crude product was purified by Biotagecolumn chromatography eluting with 5% CH₃OH: (saturated withammonia)-CH₂Cl₂, to afford the title compound as an enantiomericmixture. The mixture was separated into pure enantiomers on Prep HPLCChiral AD column eluting with 3540% Isopropanol-Hexane: 0.2% Diethylamine, to give the title compounds (13) and (13a) MS 497 (MH⁺).

Example 10 Compounds 14 and 14a

[0235]

[0236] In a similar manner as described in Example 9 above,imidazolylsodium was added to a solution of the compound (89) fromPreparative example 23, Step L, in DMF (7 ml). The reaction mixture washeated to 90° C. for 2 h then cooled and the DMF was removed. To theresidue was added saturated sodium bicarbonate and the solution wasextracted with CH₂Cl₂. The organic layer was dried over magnesiumsulfate, filtered and concentrated to dryness. The crude product waspurified by Biotage column chromatography eluting with 5% CH₃OH:(saturated with ammonia)-CH₂Cl₂, to afford the title compound as anenantiomeric mixture. The mixture was separated into pure enantiomers onPrep HPLC Chiral AD column eluting with 35-40% Isopropanol-Hexane: 0.2%Diethyl amine, to give the title compounds (14) and (14a) MS 497 (MH⁺).

EXAMPLE 11 Compound 24

[0237]

[0238] To a solution of compound 100a (1.3 g, 2.94 mmol) in CH₂Cl₂ (60mL) was added triethyl amine (1.3 mL, 9.4 mmol) and p-cyano phenylisocyanate (0.466 g, 3.24 mmol). The resulting solution was stirred atroom temperature overnight, followed by the extraction with CH₂Cl₂ andsaturated NaHCO₃. The organic layer was dried over Na₂SO₄, evaporatedand the residue purified by column chromatography, eluting with 1%-2%MeOH—NH₃/98% CH₂Cl₂ to afford compound (24) (0.870 g, 48% yield) isomer1; MS MH⁺=550.

In Vitro Plasmodium falciparum Growth Inhibition Assay

[0239] The growth activity of P. falciparum was determined by measuringthe uptake of the radiolabeled nucleic acid precursor [3H]-hypoxanthinefrom the media into infected erythrocytes by a method similar to thatreported by Jomaa et al (1999). In this method human red blood cells(RBC) were infected with P. falciparum (strain C10, partiallychloroquine resistant strain) to 0.4% parasitemia (0.4% of the RBCcontain at least 1 parasite). The infected human RBC cells were adjustedto a hematocrit of 2% and 200 μl was plated into 96 well plates. DMSOsolutions of inhibitors were added to the plates to make finalconcentrations of inhibitors from 20 μM to 0.2 μM and a finalconcentration of DMSO between 0.2 and 1%. The inhibitor-RBC solutionswere incubated for 48 hours followed by the addition of[3H]-hypoxanthine to each well and further incubated for 24 hours. Theincorporation of [3H]-hypoxanthine into the parasite DNA was measured byacid precipitating the nucleic acid content onto filters, washing thefilters and counting the incorporated radioactivity in a scintillationcounter. Controls were run to demonstrate that DMSO at levels <0.5% didnot inhibit parasite growth while a 1% DMSO solution inhibited growth30%. The ED₅₀ for fosmidomycin and chloroquine, known inhibitors of P.falciparum, were 0.6 and 0.26 μM, respectively.

[0240] Compounds of the present invention exhibit a P. falciparum ED₅₀range of between 0.05 μM and 8.6 μM.

REFERENCE

[0241] Jomaa et al, 1999, Science 285, 1573-1576. “Inhibitors of theNonmevalonate Pathway of Isoprenoid biosynthesis as Antimalarial Drugs”.

[0242] Administration and Dosage

[0243] For preparing pharmaceutical compositions from the compoundsdescribed by this invention, inert, pharmaceutically acceptable carrierscan be either solid or liquid. Solid form preparations include powders,tablets, dispersible granules, capsules, cachets and suppositories. Thepowders and tablets may be comprised of from about 5 to about 95 percentactive ingredient. Suitable solid carriers are known in the art, e.g.magnesium carbonate, magnesium stearate, talc, sugar or lactose.Tablets, powders, cachets and capsules can be used as solid dosage formssuitable for oral administration. Examples of pharmaceuticallyacceptable carriers and methods of manufacture for various compositionsmay be found in A. Gennaro (ed.), Remington's Pharmaceutical Sciences,18th Edition, (1990), Mack Publishing Co., Easton, Pa.

[0244] Liquid form preparations include solutions, suspensions andemulsions. Examples of liquid forms are water or water-propylene glycolsolutions for parenteral injection or addition of sweeteners andopacifiers for oral solutions, suspensions and emulsions. Liquid formpreparations may also include solutions for intranasal administration.

[0245] Aerosol preparations suitable for inhalation may includesolutions and solids in powder form, which may be in combination with apharmaceutically acceptable carrier, such as an inert compressed gas,e.g. nitrogen.

[0246] Also included are solid form preparations, which are intended tobe converted, shortly before use, to liquid form preparations for eitheroral or parenteral administration. Such liquid forms include solutions,suspensions and emulsions.

[0247] The compounds of the invention may be deliverable transdermally.The transdermal compositions can take the form of creams, lotions,aerosols and/or emulsions and can be included in a transdermal patch ofthe matrix or reservoir type as are conventional in the art for thispurpose.

[0248] Preferably, the compounds of the invention are administeredorally. Pharmaceutical compositions can contain any one of the compounds1-23 used in the methods of the invention in combination with one ormore additional compounds, which are useful in preventing or treatingmalaria. Additional compounds include but are not limited to the use ofquinolines (i.e., Chloroquine), folic acid antagonists (e.g.pyrimethamine), sulfonamides (e.g. sulfadiazine), antibiotics (e.g.tetracycline) and compounds which reverse Chloroquine resistance i.e.inhibitors of multidrug resistance (e.g. tetrandrine).

[0249] Preferably, the pharmaceutical preparation is in a unit dosageform. In such form, the preparation is subdivided into unit dosescontaining therapeutically effective amounts of the active component,i.e., amounts that inhibit malaria.

[0250] The compounds of this invention are administered to a patient inneed of such treatment (e.g. a mammal, such as a human being) in aneffective amount, e.g. a therapeutically effective amount, or a malariainhibitory effective amount. An effective amount is that amountnecessary to inhibit FPT, and thereby inhibit the malarial parasite.

[0251] The quantity of active compound in a unit dose of preparation maybe varied or adjusted from about 1.0 mg to about 4500 mg, preferablyfrom about 10.0 mg to about 1000 mg, according to the particularapplication.

[0252] The actual dosage employed may be varied depending upon therequirements of the patient and the severity of the condition beingtreated. Determination of the proper dosage for a particular situationis within the skill of the art. Generally, treatment is initiated withsmaller dosages which are less than the optimum dose of the compound.Thereafter, the dosage is increased by small increments until theoptimum effect under the circumstances is reached. For convenience, thetotal daily dosage may be divided and administered in portions duringthe day i.e., the total daily dosage may be divided and administered inportions up to two to four times over a 24 hour period.

[0253] The amount and frequency of administration of the compounds ofthe invention and/or the pharmaceutically acceptable salts thereof willbe regulated according to the judgment of the attending clinicianconsidering such factors as age, condition and size of the patient aswell as severity of the symptoms being treated. A typical recommendeddaily dosage regimen for oral administration can range from about 10mg/day to about 9000 mg/day, in two to four divided doses.

[0254] While the present invention has been described in conjunctionwith the specific embodiments set forth above, many alternatives,modifications and variations thereof will be apparent to those ofordinary skill in the art. All such alternatives, modifications andvariations are intended to fall within the spirit and scope of thepresent invention.

What is claimed is:
 1. A method for treating Malaria comprisingadministering to a human in need of such treatment an effective amountof a compound selected from:


2. The method of claim 1 wherein the FPT inhibitor is:


3. A method for treating malaria comprising administering to a patientin need of such treatment an effective amount of a compound used in themethod of claim 1, in combination with an effective amount of anadditional antimalaria agent and/or an additional agent for reversingantimalarial resistance.
 4. A method for treating malaria comprisingadministering to a patient in need of such treatment an effective amountof the compound used in the method of claim 2, in combination with aneffective amount of an additional antimalaria agent and/or an additionalagent for reversing antimalarial resistance.
 5. The method of claim 3,wherein said compound is administered prior to, concurrent to orsubsequent to the administration of said additional antimalaria agentand/or said agent for reversing antimalarial resistance.
 6. The methodof claim 3 wherein said additional antimalaria agent is selected fromthe group comprising: a) quinolines, b) folic acid antagonists, c)sulfonamides, and d) antibiotics.
 7. The method of claim 3 wherein saidagent for reversing antimalarial resistance is an inhibitor of multidrugresistance.
 8. The method of claim 4, wherein said compound isadministered prior to, concurrent to or subsequent to the administrationof said additional antimalaria agent and/or said agent for reversingantimalarial resistance.
 9. The method of claim 4 wherein saidadditional antimalaria agent is selected from the group comprising: a)quinolines, b) folic acid antagonists, c) sulfonamides, and d)antibiotics.
 10. The method of claim 4 wherein said agent for reversingantimalarial resistance is an inhibitor of multidrug resistance.
 11. Acompound selected from:


12. A pharmaceutical composition comprising an effective amount of acompound used in the method of claim 1, and a pharmaceuticallyacceptable carrier.
 13. A pharmaceutical composition comprising aneffective amount of the compound used in the method of claim 2, and apharmaceutical acceptable carrier.
 14. A pharmaceutical compositioncomprising an effective amount of a compound of claim 7 and apharmaceutically acceptable carrier.
 15. A pharmaceutical compositioncomprising an effective amount of (1) a compound used in the method ofclaim 1; (2) an additional antimalaria agent; and/or (3) an agent forreversing antimalarial resistance.
 16. The composition of claim 11wherein said additional antimalaria agent is selected from the groupcomprising: a) quinolines, b) folic acid antagonists, c) sulfonamides,and d) antibiotics.
 17. The composition of claim 11 wherein said agentfor reversing antimalarial resistance is an inhibitor of multidrugresistance.
 18. A pharmaceutical composition comprising an effectiveamount of (1) the compound used in the method of claim 2; (2) anadditional antimalaria agent; and/or (3) an agent for reversingantimalarial resistance.
 19. The composition of claim 14 wherein saidadditional antimalaria agent is selected from the group comprising: a)quinolines, b) folic acid antagonists, c) sulfonamides, and d)antibiotics.
 20. The composition of claim 14 wherein said agent forreversing antimalarial resistance is an inhibitor of multidrugresistance.